In cooperation with the National Flow Cytometry Resource, Los Alamos National Laboratory, Los Alamos, NM, USA, systematic investigations have been carried out using a well-established and -characterized two-step carcinogenesis model in 2-D and 3-D culture. Flow cytometric analysis of rhodamine 123 and 10N-nonyl-acridine orange staining simultaneously with the DNA stain Hoechst 33342 allowed for the determination of mitochondrial mass, activity, and function in viable, differently transformed cells as a function of location in 3-D cultures and relative to each other. A multiparameter/ multilaser flow cytometer has been used for data accquisition and the IDLYC program developed by the National Flow Cytometry Resource has been applied for data analysis. Some results have already been published: L.A. Kunz-Schughart, R.C. Habbersett, J.P. Freyer. Mitochondrial function in oncogene-transfected rat fibroblasts isolated from multicellular spheroids. Am. J. Physiol. 273: C1487-C1495, 1997. An additional manuscript in preparation will be submitted in the near future: L.A. Kunz-Schughart, R.C. Habbersett, J.P. Freyer. Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2-D and 3-D culture.